Kinetics Glossary

How fast the complex forms when analyte and ligand collide

How fast the complex falls apart

Affinity; K_D = k_d/k_a at equilibrium for a 1:1 interaction. Lower = tighter binding

K_A = k_a/k_d at equilibrium for a 1:1 interaction; equivalent to 1/K_D. Higher = tighter binding

Dissociation half-life under buffer-only conditions (no rebinding, no analyte): time for 50% of complexes to dissociate, t_½ = ln(2)/k_d

Apparent first-order rate constant from association-phase fitting. For reversible 1:1 binding k_obs = k_a·[analyte] + k_d; commonly reported in covalent and slow-binding analyses

Maximum rate of covalent bond formation between inhibitor and target once bound; the k_inact/K_i ratio is the standard covalent potency metric

Equilibrium dissociation constant of the non-covalent encounter complex preceding covalent attack; pairs with k_inact for covalent inhibitor characterization

Average time a complex stays bound; 1/k_d

SPR signal unit. 1 RU ≈ 1 pg/mm² of protein on a CM5/SA-type surface (Stenberg et al. 1991), with the exact conversion depending on surface chemistry and dn/dc. Not directly equivalent to BLI nm shifts.

BLI signal unit; measures optical thickness change. Roughly ~1 ng/mm² of protein per 1 nm shift on streptavidin biosensors, but the conversion is biosensor-chemistry dependent.

Change in refractive index per change in solute concentration. Underlies SPR/BLI mass-to-signal conversions; ~0.18–0.19 mL/g for most proteins, but glycoproteins, nucleic acids, and detergents differ.

Maximum response at saturation (all binding sites occupied)

Equilibrium response at a given concentration

Plot of response (Y) vs time (X) showing binding in real-time

The molecule immobilized on the sensor surface

The molecule in solution that flows over (SPR) or dips into (BLI)

Time period when analyte is present and binding occurs

Time period when buffer replaces analyte; complex falls apart

Signal level before/after binding; should be stable and flat

Removing bound analyte to reuse the surface for another cycle

One concentration per cycle, regenerate between. Best when regeneration is reliable

Multiple concentrations injected in sequence, no regeneration needed

Experiment format where each concentration reaches steady state before dissociation

Set of analyte concentrations (typically 2–3× dilutions) used to resolve kinetic parameters

Stepwise addition of increasing analyte concentration to measure dose-response

Covalent attachment via lysine residues (random orientation)

Non-covalent capture via His-tag on Ni-NTA surface (oriented)

High-affinity capture of biotinylated ligands

Antibody immobilized to capture ligand (e.g., anti-Fc for IgGs)

Amount of ligand immobilized (in RU or nm)

Binding limited by how fast analyte reaches the surface, not the reaction itself

Gradual increase/decrease of signal even without analyte

Sudden signal jump from refractive index difference (not true binding)

Analyte sticking to the surface or reference channel

Real biophysical enhancement of apparent affinity from multivalent interactions (e.g., bivalent IgG binding two surface-bound antigens). Becomes a fitting artifact only when 1:1 kinetic models are applied to multivalent systems — the underlying phenomenon itself is genuine.

Dissociated analyte re-associating before leaving the surface

Multiple binding populations with different kinetics

Simple, monovalent interaction. Always try this first.

1:1 Langmuir model extended with a mass-transport term. Addresses mass transport limitation (MTL): apparent k_a is depressed at low flow rates, and the response often becomes near-linear during association.

Binding followed by a stabilizing conformational change

Two independent binding sites with different kinetics

Model used when kinetics are too fast to fit directly; fit R_eq vs concentration to extract K_D. The same underlying analysis is also called "Steady-State Analysis" (Equilibrium category) and is the affinity readout from an "Equilibrium Binding" experiment format.

Analyte has two binding sites (e.g., IgG)

Sum of squared residuals between data and fit, in signal units (RU², nm²). Lower is better, and values are only comparable within a dataset of similar response amplitude. Some fitters report reduced χ²ν (χ² divided by degrees of freedom) instead.

Difference between measured data and fitted curve at each point. Should be random, not systematic

Fitting all concentrations simultaneously with shared k_a and k_d. Preferred for kinetic analysis

Fitting each concentration independently. Useful for diagnostics but not for final results

Parameters shared across curves during global fitting (e.g., k_a and k_d are linked; R_max may be local)

Uniqueness value indicating how well-determined a fitted parameter is. Close to 1 = well-determined

Standard SPR/BLI processing that combines reference subtraction (active − reference channel) with blank subtraction (sample − buffer-only injection) to remove both bulk and systematic artifacts. Double Referencing = Reference Subtraction + Blank Subtraction.

Subtracting the reference channel/surface signal from the active channel to remove bulk refractive index changes and non-specific binding. First step of double referencing.

Subtracting a buffer-only (0 nM) injection from sample injections to remove baseline drift and instrument noise. Second step of double referencing.

Zeroing the response at a defined time point so all curves start from the same level

Correcting for refractive index effects caused by DMSO or other co-solvents in small molecule assays

Processing raw sensorgrams (cropping, alignment, referencing) to prepare data for kinetic fitting

Smoothing or filtering high-frequency noise from sensorgrams without distorting kinetic features

Compensating for systematic baseline drift caused by temperature changes, surface degradation, or buffer mismatch

Plotting R_eq vs concentration to determine K_D when kinetics are too fast to fit directly

Plot of R_eq/[analyte] vs R_eq. Linear = single-site binding; curvature suggests multiple sites or cooperativity

Measure of cooperativity. n_H = 1 means no cooperativity; >1 = positive; <1 = negative

When binding of one molecule influences the affinity for subsequent molecules at other sites

Solution-phase calorimetric technique that directly measures the heat released or absorbed during binding to yield K_D, ΔH, ΔS, and stoichiometry (n) in a single experiment. The dominant commercial platforms are MicroCal PEAQ-ITC (Malvern Panalytical) and TA Instruments Affinity ITC.

Heat change upon binding. Negative ΔH = exothermic (hydrogen bonds, van der Waals)

Disorder change upon binding. Positive ΔS often indicates hydrophobic-driven interactions

Number of binding sites per molecule, typically determined by ITC

Proteolysis Targeting Chimera — a bifunctional molecule that recruits an E3 ligase to degrade a target protein

Two-component complex: PROTAC bound to either the target protein or E3 ligase alone

Three-component complex: target protein + PROTAC + E3 ligase. R_equired for degradation

Small molecule that stabilizes a protein–protein interaction, often redirecting ubiquitin ligase activity

Umbrella term for molecules (PROTACs, molecular glues) that induce targeted protein degradation

Confirmation that a compound binds its intended protein target, often measured by SPR or BLI

PROTAC ternary-complex cooperativity, defined as α = K_D(binary) / K_D(ternary). α > 1 indicates positive cooperativity (ternary tighter than binary), α < 1 indicates negative cooperativity, and α = 1 is no cooperativity.

Tool to calculate theoretical R_max from ligand level, molecular weights, and valency

Estimating required dissociation time to observe meaningful signal decay based on expected k_d

Ensuring running buffer composition matches sample buffer to minimize bulk shifts and artifacts

Testing different regeneration conditions to find one that removes analyte without damaging the ligand

Interactive guide that diagnoses common sensorgram issues and suggests corrective actions

Uploading multiple data files at once for streamlined analysis across experiments

Organizational unit in K_inetiHub that groups related experiments, fits, and reports together

SPR platform by Cytiva. Gold standard, flow cell, high sensitivity.

BLI platform family from Sartorius. Dip-and-read format covering 2/4/8/16/96 channels (Octet R2, R4, R8, RH16, R96/R96e, BLI Discovery), aimed at higher-throughput screening.

SPR imaging platform that prints up to 384 ligands on a single sensor chip via a microfluidic printhead, enabling high-throughput epitope binning and antibody characterization.

Grating-coupled interferometry (GCI) platform originally developed by Creoptix (now part of Malvern Panalytical). Label-free, low-noise, well suited to small-molecule and fragment kinetics.

Benchtop LSPR platform. Localized surface plasmon resonance, lower cost entry point.

Waveguide-based label-free technology that measures refractive index changes. Less sensitive to bulk effects than SPR.

Array-based SPR that images an entire sensor surface simultaneously, enabling high-throughput screening (e.g., Carterra)

SPR variant using nanostructured metal surfaces instead of a continuous gold film. More compact, no prism needed.

Solution-based technique that measures binding by detecting changes in thermophoretic mobility upon ligand binding. No immobilization required.

Directed movement of molecules along a temperature gradient; depends on size, charge, and hydration shell, all of which can change upon binding. In modern Monolith MST instruments the measured signal is a combination of temperature-related intensity change (TRIC) of the fluorophore plus thermophoresis ("MST = TRIC + thermophoresis"), and TRIC often dominates.

Cross-coupling between mass flux and a temperature gradient, characterized by the Soret coefficient ST. It is one component of thermophoresis (the migration response in a thermal gradient) and depends on hydration shell, surface chemistry, and size/charge — not just molecular size.

Normalized fluorescence in MST, reported as Fhot/Fcold × 1000 in per-mille units. Changes in Fnorm (ΔFnorm) upon titration are used to derive binding curves and K_D values.

Leading MST platform by NanoTemper Technologies. Capillary-based, requires fluorescent labeling or intrinsic tryptophan fluorescence.

Leading ITC instrument family, now part of Malvern Panalytical. The current platforms are PEAQ-ITC and PEAQ-ITC Automated; the older VP-ITC has been discontinued for around a decade but is still found in many established biophysics labs.